Yiyong Zhu

Adaptation of plasma membrane H+‐ATPase of rice roots to low pH as related to ammonium nutrition

The preference of paddy rice for NH(4)(+) rather than NO(3)(-) is associated with its tolerance to low pH since a rhizosphere acidification occurs during NH(4)(+) absorption. However, the adaptation of rice root to low pH has not been fully elucidated. This study investigated the acclimation of plasma membrane H(+)-ATPase of rice root to low pH. Rice seedlings were grown either with NH(4)(+) or NO(3)(-). For both nitrogen forms, the pH value of nutrient solutions was gradually adjusted to pH 6.5 or 3.0. After 4 d cultivation, hydrolytic H(+)-ATPase activity, V(max), K(m), H(+)-pumping activity, H(+) permeability and pH gradient across the plasma membrane were significantly higher in rice roots grown at pH 3.0 than at 6.5, irrespective of the nitrogen forms supplied. The higher activity of plasma membrane H(+)-ATPase of adapted rice roots was attributed to the increase in expression of OSA1, OSA3, OSA7, OSA8 and OSA9 genes, which resulted in an increase of H(+)-ATPase protein concentration. In conclusion, a high regulation of various plasma membrane H(+)-ATPase genes is responsible for the adaptation of rice roots to low pH. This mechanism may be partly responsible for the preference of rice plants to NH(4)(+) nutrition.

Role of microRNAs in plant responses to nutrient stress

BackgroundPlants must acquire at least 14 mineral nutrients from the soil to complete their life cycles. Insufficient availability or extreme high levels of the nutrients significantly affect plant growth and development. Plants have evolved a series of mechanisms to adapt to unsuitable growth conditions where nutrient levels are too low or too high. microRNAs (miRNAs), a class of small RNAs, are known to mediate post-transcriptional regulation by transcript cleavage or translational inhibition. Besides regulating plant growth and development, miRNAs are well documented to regulate plant adaptation to adverse environmental conditions including nutrient stresses.ScopeIn this review, we focus on recent progress in our understanding of how miRNAs are involved in plant response to stresses resulting from deficiency in nutrients, such as nitrogen, phosphorus, sulfur, copper and iron, as well as toxicities from heavy metal ions.ConclusionsAccumulated evidence indicates that miRNAs play critical roles in sensing the abundance of nutrients, controlling nutrient uptake and phloem-mediated long-distance transport, and nutrient homeostasis. miRNAs act as systemic signals to coordinate these physiological activities helping plants respond to and survive nutrient stresses and toxicities. Knowledge about how miRNAs are involved in plant responses to nutrient stresses promise to provide novel strategies to develop crops with improved nutrient use efficiency which could be grown in soils with either excessive or insufficient availability of nutrients.

Thermographic visualization of leaf response in cucumber plants infected with the soil-borne pathogen Fusarium oxysporum f. sp. cucumerinum.

Infection with the soil-borne pathogen Fusarium oxysporum f. sp. cucumerinum (FOC), which causes Fusarium wilt of cucumber plants, might result in changes in plant transpiration and water status within leaves. To monitor leaf response in cucumber infected with FOC, digital infrared thermography (DIT) was employed to detect changes in leaf temperature. During the early stages of FOC infection, stomata closure was induced by ABA in leaves, resulting in a decreased transpiration rate and increased leaf temperature. Subsequently, cell death occurred, accompanied by water loss, resulting in a little decrease in leaf temperature. A negative correlation between transpiration rate and leaf temperature was existed. But leaf temperature exhibited a special pattern with different disease severity on light-dark cycle. Lightly wilted leaves had a higher temperature in light and a lower temperature in dark than did in healthy leaves. We identified that the water loss from wilted leaves was regulated not by stomata but rather by cells damage caused by pathogen infection. Finally, water balance in infected plants became disordered and dead tissue was dehydrated, so leaf temperature increased again. These data suggest that membrane injury caused by FOC infection induces uncontrolled water loss from damaged cells and an imbalance in leaf water status, and ultimately accelerate plant wilting. Combining detection of the temperature response of leaves to light-dark conditions, DIT not only permits noninvasive detection and indirect visualization of the development of the soil-borne disease Fusarium wilt, but also demonstrates certain internal metabolic processes correlative with water status.

Interplay among NH4+ uptake, rhizosphere pH and plasma membrane H+-ATPase determine the release of BNIs in sorghum roots – possible mechanisms and underlying hypothesis

Aims and backgroundThe ability to suppress soil nitrification through the release of nitrification inhibitors from plant roots is termed ‘biological nitrification inhibition’ (BNI). Earlier, we reported that sorghum roots release higher BNI-activity when grown with NH4+, but not with NO3- as N source. Also for BNI release, rhizosphere pH of <5.0 is needed; beyond this, a negative effect on BNI release was observed with nearly 80% loss of BNI activity at pH >7.0. This study is aimed at understanding the inter-functional relationships associated with NH4+ uptake, rhizosphere-pH and plasma membrane H+-ATPase (PM H+-ATPase) activity in regulating the release of BNIs (biological nitrification inhibitors) from sorghum roots.MethodsSorghum was grown hydroponically and root exudates were collected from intact plants using a pH-stat system to separate the secondary acidification effects by NH4+ uptake on BNIs release. A recombinant luminescent Nitrosomonas europaea bioassay was used to determine BNI-activity. Root plasma membrane was isolated using a two-phase partitioning system. Hydrolytic H+-ATPase activity was determined. Split-root system setup was deployed to understand the localized responses to NH4+, H+-ATPase-stimulator (fusicoccin) or H+-ATPase-inhibitor (vanadates) on BNI release by sorghum.ResultsPresence of NH4+ in the rhizosphere stimulated the expression of H+-ATPase activity and enhanced the release of BNIs from sorghum roots. Fusicoccin, which stimulates H+-ATPase activity, also stimulated BNIs release in the absence of NH4+; vanadate, which suppresses H+-ATPase activity, also suppressed the release of BNIs. NH4+ levels (in rhizosphere) positively influenced BNIs release and root H+-ATPase activity in the concentration range of 0-1.0 mM, indicating a close relationship between BNI release and root H+-ATPase activity with a possible involvement of carrier-mediated transport for the release of BNIs in sorghum.ConclusionOur results suggest that NH4+ uptake, PM H+-ATPase activity, and rhizosphere acidification are functionally inter-connected with BNI release in sorghum. Such knowledge is critical to gain insights into why BNI function is more effective in light-textured, mildly acidic soils compared to other soil types.

Genome-wide identification of phosphate-deficiency-responsive genes in soybean roots by high-throughput sequencing

AimsSoybean is an important food crop as well as a promising energy source. Because soybean is self-sufficient in nitrogen, phosphorus, in the orthophosphate form (Pi), becomes the most limiting macronutrient affecting the growth and productivity of soybean, especially in acidic and alkaline soils. It has been documented that plants have developed a series of physiological and biochemical strategies to adapt to Pi deficiency, but the mechanistic details of soybean response to Pi deficiency, especially those at the molecular level, are largely unknown. In this study, we aim to understand how soybean plants respond to Pi deficiency in soils by identifying and analysing Pi-responsive genes in the roots of soybean at the whole-genome scale.MethodsThe transcriptome in soybean roots under Pi-deficiency was analyzed using the Illumina’s digital gene expression (DGE) high-throughput sequencing platforms, and the expression profiles of arbitrarily selected Pi-responsive genes identified in the current research were validated by quantitative RT-PCR.ResultsA total of 1612 genes were found to be differentially expressed in soybean roots after Pi deficiency for seven days; 727 genes were up-regulated, and 885 genes were down-regulated. Gene ontology (GO) enrichment analysis showed that 17 GO terms of biological processes were significantly enriched including photosynthesis, iron ion transport, dUTP metabolism, cell wall organization, fatty acid metabolism and stress responses. Genes possibly involved in regulating Pi homeostasis, nutrient uptake and transport, homeostasis control of reactive oxygen species, calcium signaling, hormonal signaling and gene transcription were included in the differentially expressed genes. Quantitative RT-PCR was used to analyze the expression of 30 arbitrarily selected genes and 29 of them were confirmed to exhibit similar differential expression patterns under Pi deficiency as revealed by the high throughput DGE sequencing.ConclusionsThese results provide useful information for identifying and characterizing important components in the Pi signaling network in soybean and enhance understanding of the molecular mechanisms by which plants adapt to low Pi stress.

Involvement of Plasma Membrane H+-ATPase in Adaption of Rice to Ammonium Nutrient

The preference of paddy rice for NH4+ rather than NO3ˉ is associated with its tolerance to low pH since a rhizosphere acidification occurs during NH4+ absorption. However, the adaptation of rice root to low pH has not been fully elucidated. The plasma membrane H+-ATPase is a universal electronic H+ pump, which uses ATP as energy source to pump H+ across the plasma membranes into the apoplast. The key function of this enzyme is to keep pH homeostasis of plant cells and generate a H+ electrochemical gradient, thereby providing the driving force for the active influx and efflux of ions and metabolites across the plasma membrane. This study investigated the acclimation of plasma membrane H+-ATPase of rice root to low pH. This mechanism might be partly responsible for the preference of rice plants to NH4+ nutrition. Key words: rice; ammonium nutrient; plasma membrane H+-ATPase

Analysis of EF-Hand Proteins in Soybean Genome Suggests Their Potential Roles in Environmental and Nutritional Stress Signaling

Calcium ion (Ca2+) is a universal second messenger that plays a critical role in plant responses to diverse physiological and environmental stimuli. The stimulus-specific signals are perceived and decoded by a series of Ca2+ binding proteins serving as Ca2+ sensors. The majority of Ca2+ sensors possess the EF-hand motif, a helix-loop-helix structure which forms a turn-loop structure. Although EF-hand proteins in model plant such as Arabidopsis have been well described, the identification, classification, and the physiological functions of EF-hand-containing proteins from soybean are not systemically reported. In this study, a total of at least 262 genes possibly encoding proteins containing one to six EF-hand motifs were identified in soybean genome. These genes include 6 calmodulins (CaMs), 144 calmodulin-like proteins (CMLs), 15 calcineurin B-like proteins, 50 calcium-dependent protein kinases (CDPKs), 13 CDPK-related protein kinases, 2 Ca2+- and CaM-dependent protein kinases, 17 respiratory burst oxidase homologs, and 15 unclassified EF-hand proteins. Most of these genes (87.8%) contain at least one kind of hormonal signaling- and/or stress response-related cis-elements in their -1500 bp promoter regions. Expression analyses by exploring the published microarray and Illumina transcriptome sequencing data revealed that the expression of these EF-hand genes were widely detected in different organs of soybean, and nearly half of the total EF-hand genes were responsive to various environmental or nutritional stresses. Quantitative RT-PCR was used to confirm their responsiveness to several stress treatments. To confirm the Ca2+-binding ability of these EF-hand proteins, four CMLs (CML1, CML13, CML39, and CML95) were randomly selected for SDS–PAGE mobility-shift assay in the presence and absence of Ca2+. Results showed that all of them have the ability to bind Ca2+. This study provided the first comprehensive analyses of genes encoding for EF-hand proteins in soybean. Information on the classification, phylogenetic relationships and expression profiles of soybean EF-hand genes in different tissues and under various environmental and nutritional stresses will be helpful for identifying candidates with potential roles in Ca2+ signal-mediated physiological processes including growth and development, plant-microbe interactions and responses to biotic and abiotic stresses.

Further insights into underlying mechanisms for the release of biological nitrification inhibitors from sorghum roots

BackgroundSorghum roots release two categories of biological nitrification inhibitors (BNIs) – hydrophilic-BNIs and hydrophobic-BNIs. Earlier research indicated that rhizosphere pH and plasma membrane (PM) H+ATPase are functionally linked with the release of hydrophilic BNIs, but the underlying mechanisms are not fully elucidated. This study is designed to reveal further insights into the regulatory mechanisms of BNIs release in root systems, using three sorghum genetic stocks.MethodsSorghum plants were grown in a hydroponic system with pH of nutrient solutions ranging from 3.0 ̴ 9.0. Pharmacological agents [(fusicoccin and vanadate) and anion-channel blockers (−niflumic acid (NIF) and anthracene-9-carboxylate (A9C)] were applied to root exudate collection solutions; BNI activity was determined with luminescent Nitrosomonas europaea bioassay. Sorgoleone levels in root exudates and H+ excretion from roots were determined. Two-phase partitioning system is used to isolate root plasma membrane (PM) and H+ ATPase activity was determined.ResultsA decrease in rhizosphere pH improved the release of hydrophilic-BNIs from roots of all the three sorghum genotypes, but had no effect on the release of hydrophobic-BNIs. Hydrophobic-BNI activity and sorgoleone levels in root-DCM wash are positively correlated. Fusicoccin promoted H+extrusion and stimulated the release of hydrophilic-BNIs. Vanadate, in contrast, suppressed H+ extrusion and lowered the release of hydrophilic-BNIs. Anion-channel blockers did not inhibit the release of hydrophilic BNIs, but enhanced H+-extrusion and hydrophilic-BNIs release.ConclusionRhizosphere pH has a major influence on hydrophilic-BNIs release, but not on the release of hydrophobic-BNIs. The low rhizosphere pH stimulated PM-H+ ATPase activity; H+-extrusion is closely coupled with hydrophilic-BNIs release. Anion-channel blockers stimulated H+ extrusion and hydrophilic-BNIs release. Our results indicate that some unknown membrane transporters are operating the release of protonated BNIs, which may compensate for charge balance when transport of other anions is suppressed using anion-channel blockers. A new hypothesis is proposed for the release of hydrophilic-BNIs from sorghum roots.

Early Transcriptomic Response to Phosphate Deprivation in Soybean Leaves as Revealed by RNA-Sequencing

Low phosphate (Pi) availability is an important limiting factor affecting soybean production. However, the underlying molecular mechanisms responsible for low Pi stress response and tolerance remain largely unknown, especially for the early signaling events under low Pi stress. Here, a genome-wide transcriptomic analysis in soybean leaves treated with a short-term Pi-deprivation (24 h) was performed through high-throughput RNA sequencing (RNA-seq) technology. A total of 533 loci were found to be differentially expressed in response to Pi deprivation, including 36 mis-annotated loci and 32 novel loci. Among the differentially expressed genes (DEGs), 303 were induced and 230 were repressed by Pi deprivation. To validate the reliability of the RNA-seq data, 18 DEGs were randomly selected and analyzed by quantitative RT-PCR (reverse transcription polymerase chain reaction), which exhibited similar fold changes with RNA-seq. Enrichment analyses showed that 29 GO (Gene Ontology) terms and 8 KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways were significantly enriched in the up-regulated DEGs and 25 GO terms and 16 KEGG pathways were significantly enriched in the down-regulated DEGs. Some DEGs potentially involved in Pi sensing and signaling were up-regulated by short-term Pi deprivation, including five SPX-containing genes. Some DEGs possibly associated with water and nutrient uptake, hormonal and calcium signaling, protein phosphorylation and dephosphorylation and cell wall modification were affected at the early stage of Pi deprivation. The cis-elements of PHO (phosphatase) element, PHO-like element and P responsive element were present more frequently in promoter regions of up-regulated DEGs compared to that of randomly-selected genes in the soybean genome. Our transcriptomic data showed an intricate network containing transporters, transcription factors, kinases and phosphatases, hormone and calcium signaling components is involved in plant responses to early Pi deprivation.

Overexpression of Phosphate Transporter Gene CmPht1;2 Facilitated Pi Uptake and Alternated the Metabolic Profiles of Chrysanthemum Under Phosphate Deficiency

Low availability of phosphorus (P) in the soil is the principal limiting factor for the growth of cut chrysanthemum. Plant phosphate transporters (PTs) facilitate acquisition of inorganic phosphate (Pi) and its homeostasis within the plant. In the present study, CmPht1;2 of the Pht1 family was cloned from chrysanthemum. CmPht1;2 is composed of 12 transmembrane domains and localized to the plasma membrane. Expression of CmPht1;2 in roots was induced by Pi starvation. Chrysanthemum plants with overexpression of CmPht1;2 (Oe) showed higher Pi uptake, as compared to the wild type (WT), both under Pi-starvation and Pi-sufficient conditions, and also showed a higher root biomass compared to WT in the Pi-starvation conditions. Seven days after the P-deficiency treatment, 85 distinct analytes were identified in the roots and 27 in the shoots between the Oe1 plant and WT, in which sophorose, sorbitol (sugars), hydroxybutyric acid (organic acids), and ornithine (amino acid) of CmPht1;2 overexpressing chrysanthemum are specific responses to P-starvation.